This invention is directed to improved methods for the extraction of cocoa procyanidin monomers and oligomers from the cocoa solids.
It is known that regular consumption of dietary polyphenols, commonly found in a variety of fruits and vegetables, is beneficial. Red wine, green tea and cocoa have all been identified as being rich in polyphenols, and the regular consumption red wine and green tea have both been shown to be inversely associated with heart disease deaths in industrialized countries.
It is well-known that the polyphenols of cocoa contribute significantly to the development of flavour in the fermented and roasted cocoa bean. Astringent and bitter flavors in cocoa have been traditionally associated with the presence of xanthine alkaloids and polyphenols in the cocoa beans. For this reason, various methods have been developed over the years to extract the cocoa polyphenols to verify their presence, to quantify their amounts, and to identify them. The cocoa polyphenols are primarily cocoa procyanidins. However, no extraction method has thus far been optimized to yield extracts high in cocoa procyanidins.
It is now known that the cocoa procyanidin oligomers show a clear relationship between structure and function, meaning that individual oligomers, or fractions containing several oligomers of a similar size, show specific biological functions which are not affected by other oligomers. Thus, it is important to ensure that the extraction procedure utilized not only results in the highest possible solubilization of the cocoa polyphenols, but that it is effective at extracting all the cocoa polyphenol oligomers present in the bean.
The extraction of cocoa beans using water or an organic solvent, or a mixture of water and an organic solvent, has been used to remove the xanthine alkaloids (predominantly caffeine and theobromine) and other soluble constituents of the cocoa bean which impart a bitter, disagreeable flavor. Included among these bitter-tasting soluble constituents are the procyanidins.
U.S. Pat. No. 1,750,795 (issued to Defren in 1926), discloses a process for removing the xe2x80x9cgreater part of the soluble bitter constituents of the beansxe2x80x9d by soaking the beans in water at 60xc2x0 C. and then discarding the water and roasting the beans.
Hot water treatments have been used to remove the xanthines in order to provide stimulant-free cocoa beans. See U.S. Pat. No. 4,407,834 xe2x80x9cDetheobromination of Cocoaxe2x80x9d (issued to Chiovini et al., Jun. 28, 1983) and U.S. Pat. No. 4,755,391 xe2x80x9cRemoval of Methylxanthines from Cacao Materialsxe2x80x9d (issued to Chiovini et al, Jul. 5, 1988).
Cocoa extracts have been prepared by extracting cocoa solids, prepared from fermented, conventionally roasted cocoa beans or cocoa nibs, with water and/or alcohols. Osakabe et al. used ethanol, either absolute or aqueous at greater than 40% v/v with deionized water at ambient temperature to prepare a crude polyphenol extract. See JP 946-64717 xe2x80x9cFood or Beverage Product for Preventing Gastric Ulcersxe2x80x9d (Oct. 4, 1995).
Zieglader et al. used methanol to extract the polyphenols from cocoa beans at ambient temperature. See xe2x80x9cAntioxidative Effects of Cocoaxe2x80x9d (Rev. Choc.Confect. Bak, 8:3-6, 1983) which discloses the preparation of a methanol extract. The extract contained xe2x80x9cmonomer tannin precursors (catechins, anthocyanidins and their soluble condensates)xe2x80x9d and that is used as an additive for oil to preserve it from oxidation. Griffiths et al. used methanol extracts, again obtained at ambient temperatures, to obtain polyphenol extracts from ripe cocoa nibs, which were used to study the characterization of plant polyphenols in cocoa and other plants. See xe2x80x9cA Comparative Study of the Seed Polyphenols of the Genus Theobromaxe2x80x9d, (Biochemical J. 74: 362-365, 1960). Rigaud et al. made an extract from lyophilized cocoa beans and grape seeds, and noted that the use of methanol as a solvent precluded the presence of the higher oligomers. See xe2x80x9cNormal-Phase High-Performance Liquid Chromatographic Separation of Procyanidins from Cacao Beans and Grape Seedsxe2x80x9d, (J. Chromatography 654:255-60, 1993). Jalal and Collin prepared extracts from different parts of the cocoa plant in order to analyze the polyphenols present in each part of the plant. The extraction was carried out using 70% cold methanol, followed by ethyl acetate. See xe2x80x9cPolyphenols of Mature Plant, Seedling and Tissue Cultures of Theobroma Cacaoxe2x80x9d (Phytochemistry, 16:1377-1380, 1977).
Acetone/water has also be used for cocoa bean extractions. Clapperton et al. report the extraction of defatted cocoa powder made from fermented cocoa beans using cold 70% acetone. See xe2x80x9cPolyphenols and Cocoa Flavor, Groupe Polyphenols,xe2x80x9d (XVIth Intern. Conf., Lisbon, Portugal, Jul. 13-16, 1992). Rigaud combines a first extraction with ethanol with a second extraction using a 60% acetone/water mixture. See xe2x80x9cNormal-Phase High-Performance Liquid Chromatographic Separation of Procyanidins from Cacao Beans and Grape Seedsxe2x80x9d (J.
Chromatography, 654:255-60, 1993).
Traditionally, cocoa beans are treated and processed in such a way as to minimize the bitter taste which the polyphenols impart to the beans, and this results in a reduction of the polyphenol content of the bean. The two ways in which the polyphenol content is significantly reduced are fermentation of cocoa beans in their husks and roasting the fermented cocoa beans in order to crack their husks and aid the de-hulling process. Zieglader at al. report a loss of antioxidant activity in extracts from cocoa beans which are fermented compared to extracts from unfermented beans, and correlate this reduction in antioxidant potential with a reduced amount of polyphenols in the extracts of fermented beans. All the previously discussed extraction methods were carried out using fermented beans, and in many cases the beans were also roasted. Therefore, the procyanidin yields are much lower than those found in extracts from unfermented beans.
It has been a common practice to follow an initial solvent extraction step with an ethyl acetate extraction step. See Forsyth and Roberts, xe2x80x9cCacao Polyphenolic Substances: 3. The Structure of Cacao Leucocyanidin 1xe2x80x9d Biochem. J. 74, 374-378, 1960; Thompson et al., xe2x80x9cPlant Procyanidins. Part I. Introduction; the Isolation, Structure and Distribution in Nature of Plant Procyanidinsxe2x80x9d J. Chem. Soc. Perkin I, Vol. 11, 1387-99, 1972; Jalal and Collins, xe2x80x9cPolyphenols of Mature Plant, Seedling and Tissue Cultures of Theobroma Cacaoxe2x80x9d, Phytochemistry, 16:1377-1380, 1977, Porter et al. xe2x80x9cFlavans and Proanthocyanidinsxe2x80x9d Chapter Two in xe2x80x9cThe Flavonoidsxe2x80x9d, Ed., J. B. Harborne, Chapman and Hall Ltd., London, 1988; U.S. Pat. No. 5,554,645 (issued Oct. 3, 1994 to Romanczyk et al). This has the effect of producing an extract which contains the monomers and lower oligomers and none, or very little, of the higher oligomers. See Lea A. xe2x80x9cThe Phenolics of Ciders: Oligomeric and Polymeric Procyanidinsxe2x80x9d, J. Sci. Fd Agric. 29, 471-477, 1978.
Whether or not the cocoa beans are defatted prior to extraction has an impact upon the yield of polyphenols extracted. If the beans are not defatted, the fat in the beans interferes with the solubilizing activity of the solvent, and the polyphenol yields are considerably reduced (Lazarus et al., xe2x80x9cFlavonoids and Other Polyphenolsxe2x80x9d in Methods in Enzymology series. Edited by Lester Packer, Academic Press, New York, in press).
Cocoa extracts have been prepared from cocoa solids prepared from unfermented or fermented sun dried cocoa beans. The beans were ground, defatted and extracted. A 70% acetone/30% deionized water mixture was used, followed by a 70% methanol extraction and two chloroform extractions. Then follows an extraction with ethyl acetate, the addition of water, and the removal of the ethyl acetate. Alternatively, the beans were extracted with 70% acetone. In both cases the extractions were carried out at room temperature and the aqueous extracts were freeze-dried. See U.S. Pat. No. 5,554,645 (issued Oct. 3, 1994 to L. Romanczyk et al.).
Furthermore, cocoa extracts can be prepared from partially defatted cocoa solids that are prepared from cocoa beans which have not been roasted. The resulting cocoa solids, whether prepared from fermented, underfermented, or unfermented cocoa nibs, have a higher cocoa polyphenol content, i.e., cocoa procyanidin content, than cocoa solids prepared from conventionally roasted cocoa beans or cocoa nibs. See U.S. Pat. No. 6,015,913 (issued Jan. 18, 2000 to K. S. Kealey et al.).
Defatted, freeze-dried, unfermented cocoa beans contain about 2% xanthine alkaloids, and traditionally processed beans contain larger amounts. Theobromine is a degradation product of caffeine, and both are well-known stimulants of the nervous system. Excessive xanthine alkaloid intake is not thought to be beneficial. Hence, in some cases, it may be desirable to cocoa extracts free of the xanthine alkaloids.
Thus, there is a need for an improved process to extract cocoa procyanidins from cocoa solids, and, in some cases, to decaffeinate and detheobrominate the cocoa extracts.
The present invention provides an improved method for extracting cocoa polyphenols including cocoa procyanidins from partially or fully defatted cocoa solids prepared from non-roasted cocoa beans. The improved process comprises a single extraction, at atmospheric pressure or under pressure, with a solvent selected from the group consisting of an acidified organic solvent capable of solubilizing the polyphenols, or aqueous mixtures thereof. An edible acid is added to solvent or aqueous-solvent mixture in an amount sufficient to lower the pH from about 6.5 to about 2 to about 4. The cocoa solids are prepared from unfermented or underfermented cocoa beans. The organic solvent may be selected from the group consisting of lower alkyl alcohols, lower alkyl ketones, and lower alkyl acetate. Suitable solvents include methanol, ethanol, isopropanol, acetone, methyl acetate, or ethyl acetate. The aqueous solvent mixture preferably contains up to about 50% water by volume. Preferred aqueous-solvent mixtures include isopropanol and about 50-70% water, isopropanol and about 20% water which preferentially extracts monomers and oligomers up to and including hexamers, acetone and about 80% to about 100% water, acetone and about 50% to about 70% water, or 100% ethanol which preferentially extracts cocoa procyanidin oligomers higher than the heptamers is enhanced.
The present invention also provides a method of extracting cocoa polyphenols including cocoa procyanidin monomers and oligomers from partially defatted or fully defatted cocoa solids prepared from cocoa beans that have not been roasted. The method comprises the step of extracting, at atmospheric pressure or under pressure, the cocoa solids with a non-acidified organic solvent capable of solubilizing cocoa polyphenols or non-acidified aqueous mixtures thereof. Preferably, the cocoa beans are unfermented or underfermented cocoa beans. The beans typically have a fermentation factor of 275 or less. The preferred beans include slaty, purple, or purple brown cocoa beans, or mixtures of slaty and purple cocoa beans, purple and brown cocoa beans, or slaty, purple and brown cocoa beans. The unfermented or underfermented cocoa beans contain at least about 1% up to about 15% by weight of total cocoa procyanidins per gram of defatted cocoa solids, typically about 4-7%. The organic solvent may be selected from the group consisting of a lower alkyl alcohol, a lower alkyl ketone, and a lower alkyl acetate. Suitable organic solvents are methanol, ethanol, isopropanol, acetone, methyl acetate, or ethyl acetate. The aqueous mixture can contain up to about 50% water by volume. Preferred solvents include 50-60% acetone, and 100% ethanol. The extraction can be carried out at a temperature from 0xc2x0 C. up to the boiling point of the solvent or aqueous solvent mixture used at the extraction pressure used. When the solvent is methanol, the extraction is preferably carried out at room temperature. When the solvent is ethanol, the extraction is preferably carried out at about 20xc2x0 C. to about 50xc2x0 C. When the solvent is isopropanol, the extraction is preferably carried out at about 70xc2x0 C. When the solvent is an aqueous-acetone mixture, the extraction is preferably carried out at about 50xc2x0 C. up to the boiling point of the mixture.
In the above process, when an acidified solvent or solvent mixture is used, a lower extraction temperature is preferred. When a non-acidified solvent or solvent mixture is used, a higher extraction temperature can be used.
The present invention also provides a method for selectively extracting low molecular weight cocoa procyanidin oligomers from partially or filly defatted cocoa solids, prepared from cocoa beans that have not been roasted. The solvent used is a solvent which preferentially extracts the lower oligomers, e.g., methyl acetate or ethyl acetate. Preferably, the cocoa beans are unfermented or underfermented cocoa beans. The extraction is preferably carried out at about 20xc2x0 C. to about 50xc2x0 C. The resulting cocoa extracts consist essentially of monomers, dimers and trimers.
The present invention also provides a method for selectively extracting higher molecular weight cocoa procyanidin oligomers from partially defatted or filly defatted cocoa solids prepared from cocoa beans that have not been roasted. The method comprises the steps of (a) extracting the cocoa solids with ethyl acetate; (b) recovering the extracted cocoa solids; (c) extracting the recovered extracted cocoa solids with a solvent which is a good solvent for the higher oligomer, e.g., acetone and ethanol and mixtures there of with up to 50% water; (d) separating the cocoa solids from the cocoa extract; and (e) optionally drying the cocoa extract. The extraction is preferably carried out at about 20xc2x0 C. to about 50xc2x0 C. Preferably the cocoa beans are unfermented or underfermented. The cocoa extracts consists essentially of at least tetramers and higher oligomers.
The present invention further provides a continuous method for extracting, at atmospheric pressure or under pressure, cocoa polyphenols including cocoa procyanidins from partially defatted or fully defatted cocoa solids using an organic solvent suitable for solubilizing cocoa procyanidins, or mixtures thereof with water. The process comprises the steps of (a) contacting a series of cells containing the cocoa solids with a solvent flowing in a counter-current direction, (b) recovering the cocoa extract, and (c) drying the cocoa extract. Preferred solvents include lower alkyl alcohols, lower alkyl ketones, and lower alkyl acetates. Suitable lower alkyl alcohols are methanol, ethanol, and isopropanol. A suitable lower alkyl ketones is acetone. Suitable lower alkyl acetates are methyl acetate and ethyl acetate. Preferably, the cocoa extract is recycled until substantially no more cocoa procyanidins are extracted from the cocoa solids. When the solvent is the organic solvent, the cocoa extract is dried by flashing off the solvent. When the solvent is the aqueous-organic mixture, the cocoa extract is dried by flashing off the solvent before drying the aqueous extract. The aqueous extracts can be dried by freeze drying, spray drying, flash drying, or drum drying.
The present invention provides a method for recovering cocoa procyanidins and theobromine from partially defatted or fully defatted cocoa solids, prepared from cocoa beans. The process comprises the steps of (a) extracting the cocoa solids with ethanol at about 50xc2x0 C. to about 70xc2x0 C., (b) chilling the ethanol extract at a temperature and for a time sufficient to precipitate out the theobromine, (c) separating the precipitated out theobromine, and (d) removing the ethanol from extract to recover the dried cocoa procyanidins. Preferably, the beans are unfermented or underfermented cocoa beans that have not been roasted. The present invention also provides a method of minimizing the amount of theobromine in the cocoa procyanidins extracted from partially defatted or fully defatted cocoa solids, prepared from unroasted, preferably unfermented or underfermented, cocoa beans. The method comprises the steps of (a) extracting the cocoa solids with isopropanol to produce an alcoholic extract containing cocoa procyanidins and theobromine, (b) chilling the alcoholic extract at a temperature and for a time sufficient to precipitate out the theobromine, and (c) separating the theobromine from the alcoholic extract. In the above processes, the chilling is carried out for up to about 12 hours at 0xc2x0 C. to about 5xc2x0 C. and the separating step is carried out by filtering the extract, centrifuging the extract, or by an adsorbing the theobromine on a suitable absorbent.